Endometriosis, like cancer, has also been shown to be characterized by increased proliferation, invasion and angiogenesis; however, the specific mechanisms underlying this estrogen-dependent, invasive disease are still unknown. The two main cellular components of the endometrium, namely epithelial (EEC) and stromal (HESC) cells, show a differential expression of CXCR4 and CXCL12 proteins: endometrial epithelial cells express higher CXCR4 levels than stromal cells; conversely, endometrial stromal cells expressed higher levels of CXCL12 as previously reported from others and our laboratory. Also, we showed that CXCR4 expression is differentially regulated by estradiol (E2) and progesterone (P4) in endometrial cell lines. Finally, we have also shown that CXCR4 is significantly overexpressed in the lesions of the rat model of endometriosis as well as in human endometriotic tissues. The expression of CXCR4 protein in the endometriotic lesions was significantly higher compared to secretory control endometrium, which expresses the highest expression of this chemokine receptor. Based on these preliminary observations, we hypothesize that activation of the CXCR4-CXCL12 axis is a key step in the development and progression of endometriosis. Specifically, this proposal aims to investigate the role of CXCR4 signaling induced by its ligand, CXCL12, in promoting cell proliferation, invasion, and angiogenesis in endometriosis. The main hypothesis driving this proposal is that overexpression of CXCR4 protein in endometriotic epithelial cells reaching the peritoneal cavity in the menstrual fluid, and its unchartered activation by CXCL12, stimulates proliferation, invasion, and angiogenesis leading to lesion establishment. To test these hypotheses, first the functionality of the CXCR4-CXCL12 axis in inducing proliferation as well as invasion/migration mechanisms will be assessed in the context of endometrial and endometriotic cells (Specific Aim #1). Next, the molecular mechanisms activated by CXCL12 binding to CXCR4 in these cellular contexts will be determined (Specific Aim #2). In Specific Aim #3, the efficacy of the CXCR4 inhibitor CTCE-9908 in preventing the development of endometriosis will be assessed in vivo using a well-validated rat model of endometriosis. We speculate that stromal cells in the endometriotic tissue produce CXCL12 in a paracrine manner, thus creating a microenvironment inducive to survival of CXCR4-expressing epithelial cells. The knowledge that will be gained from this project is important to understand how endometrial cells are able to proliferate, invade and survive in ectopic sites and how the inflammatory reaction can provide the appropriate cell environment to induce endometrial cell survival at ectopic locations. Also, these studies will provide insights fo the development of future therapeutic modalities for this incurable condition targeting the CXCR4-CXCL12 axis. PUBLIC HEALTH RELEVANCE: Endometriosis affects millions of women during their reproductive life causing pain and infertility. No cure is available, and treatments are limited. This disease is characterized by inflammation in the pelvic area. This study will investigate the role of an inflammatory molecule, CXCR4, in the development of endometriosis and whether blocking its action could represent a novel treatment for this disease.